In the description which follows, references are made to certain literature citations which are listed at the end of the specification.
Many bacteria produce and secrete zinc metalloproteases. For example, Pseudomonas aeruginosa produces at least two zinc metalloproteases, elastase and alkaline protease. Elastase degrades several important biological substances including elastin, immunoglobulins, collagen, transferrin and complement components (1). Alkaline protease has been shown to degrade C1q and C3 proteins of serum complement (2) and gamma interferon (3) P. aeruginosa also secretes Las A protease, which has both elastolytic and staphylolytic activity and has some properties of a metalloprotease (4-6).
Burkholderia (Pseudomonas) cepacia produces both a 36 kDa zinc metalloprotease (PSCP) which is immunologically related to elastase and a related 40 kDa protease (7).
Bacillus thermoproteolyticus secretes thermolysin, a heat-stable neutral zinc metalloprotease. There is 28% sequence homology between P. aeruginosa elastase and thermolysin (8), with greater homology in certain regions, particularly around the active site. However, comparison of the three-dimensional structures of thermolysin and P. aeruginosa elastase reveals a striking similarity (9).
Vibrio cholerae secretes a 33 kDa zinc metalloprotease, HA/protease, (10) which can cleave biologically important substrates such as mucin, fibronectin and bactoferritin (11). Hase and Finkelstein (12,13) have demonstrated that the V. cholerae HA/protease is related to P. aeruginosa elastase.
The bacterial metalloproteases have been shown to contribute to the virulence of many pathogenic organisms (14) which cause serious health problems.
For example, pulmonary dysfunction as a result of chronic airway infection is responsible for the vast majority of deaths in cystic fibrosis (CF) patients (15). Despite advances in microbial therapy, the treatment and prevention of infections due to P. aeruginosa and B. cepacia remains a clinical challenge (15). Although major efforts are underway to develop gene therapy as a treatment for CF patients, the practical applications of gene therapy and potential for success may not be determined for some time. Other avenues of infection control continue to be an important area for investigation.
P. aeruginosa has been reported to be present in 60% of respiratory tract cultures from CF patients and once colonization has occurred, P. aeruginosa is difficult or impossible to eradicate (16). A large array of virulence factors have been identified and shown to contribute to the pathogenesis of P. aeruginosa infections. These include elastase, alkaline protease, exotoxin A, exoenzyme S, pyochelin, pyoverdin, phospholipase C, pili, outer membrane proteins, lipopolysaccharide (LPS) and alginate.
B. cepacia is a nosocomial pathogen which has been isolated with increasing frequency from respiratory infections in CF patients over the past 20 years (15). Acquisition of B. cepacia can pose particular problems because of its resistance to many anti-pseudomonal antibiotics. For example, in one study of 55 CF patients with B. cepacia pulmonary infection, 39 (70%) had acquired multiresistant strains (17). Once acquired, B. cepacia is nearly impossible to eradicate.
The majority of strains (90%) of B. cepacia are protease positive (18). Proteases appear to be the major extracellular virulence factors produced by B. cepacia. McKevvit et al. (19) isolated a zinc metalloprotease, designated PSCP, which was produced by 90% of B. cepacia CF isolates. This protease was shown to degrade casein, gelatin, collagen, but not elastin and to cause bronchopneumonia when instilled intratracheally into rats (19).
Besides having a direct role in tissue destruction and injury, bacterial proteases such as elastase can function to modulate the host immune system and assist the bacterium in evading host defences (20-22).
Numerous studies since the late 1970's have analyzed the presence of serum antibodies to several P. aeruginosa antigens in CF patients (23-27). These studies all conclude that CF patients make antibodies to a variety of P. aeruginosa antigens and that elevated titres generally correlate with severity of disease.
Although CF patients produce high levels of antibodies to P. aeruginosa, several studies have indicated that these antibodies are not effective in clearance of the organism from the lungs.
These studies indicate that there remains a need for therapeutic strategies to improve the production of effective antibodies to combat infections with organisms such as P. aeruginosa and B. cepacia.